Many higher animals have five different classes of immunoglobulins, IgG, IgA, IgM, IgD, and IgE. Each immunoglobulin class differs in properties such as size, charge, amino acid composition, and sugar content. Of these classes, IgM accounts for approximately 10% of all plasma immunoglobulins. IgM is the major component of early antibodies produced against cell-membrane antigens, infectious microorganisms, or soluble antigens, which have a complex antigenicity.
Human IgMs usually have a pentameric structure. Each of the five subunits constituting this pentameric structure has a four-stranded structure similar to that of IgG. The amino acid sequence of the μ chain, which is the heavy chain of IgM, is different from that of the γ chain, which is the heavy chain of IgG. The following differences can also be seen:                The μ chain has an extra constant domain than the γ chain.        The μ chain has four more oligosaccharide chains than the γ chain.        
IgM has a polypeptide chain called the J chain, which is not found in IgG. The J chain is considered to assist the association of μ chains prior to secretion of IgM from antibody producing cells.
With advances in monoclonal antibody technology and recombinant DNA technology, large-scale production of pure immunoglobulins has become possible in recent years. Furthermore, gene recombination techniques have enabled production of chimeric antibodies and humanized antibodies. Chimeric antibodies are antibodies having a structure in which the variable regions have been replaced with variable regions derived from a different species. For example, “chimeric antibodies” comprising variable regions of non-human antibodies and the constant regions of human antibodies (Non-Patent Document 1/Proc. Natl. Acad. Sci. U.S.A., (1984) 81:6851) are known. Also known are humanized antibodies in which the complementarity determining regions (CDR) of other animal species are transferred into human immunoglobulins (Non-Patent Document 2/Nature (1986) 321:521).
Actual examples of antitumor antibodies are the anti-CD20 human chimeric antibody RITUXAN® (IDEC), and the anti-HER2/neu humanized antibody HERCEPTIN® (Genentech), which have completed clinical trials and have already been approved. These antibodies are now commercially available. Antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC) activity and complement-dependent cytotoxicity (hereinafter referred to as CDC) activity are known as effector functions of IgG and IgM. Since IgM has a higher CDC activity compared to IgG, it has an extremely high chance of becoming an anti-tumor antibody having CDC activity as its main effect. However, as described above, unlike IgG, IgM forms a multimer. Therefore, industrial scale production of recombinant IgM had been considered difficult.
IgM is also very unstable compared to IgG and has a low solubility. Therefore, the production of a highly concentrated and stable IgM solution is difficult. For example, Cytotherapy, 2001, 3(3), 233-242 (Non-Patent Document 5) reports that, even when IgM had been stored at −20° C., precipitation and decrease of activity occurred upon thawing. Furthermore, according to the report, IgM easily aggregates and precipitates during storage. It was especially difficult to ensure an IgM stability sufficient enough to withstand pharmaceutical use only through optimization of pH and buffer type.
Therefore, various attempts are being made to stabilize antibodies by methods other than optimization of pH and buffer type. For example, WO 2002/096457 (Patent Document 1) discloses formulations for stabilizing highly concentrated antibodies that comprise acidic ingredients. This method uses MgCl2 and CaCl2 as additives to stabilize the antibodies, but the stabilization is carried out to prepare IgG formulations, and IgM formulations are not mentioned. As described above, unlike IgG, IgM exists as a multimer, and unlike intrinsically stable IgG, IgM readily aggregates. Therefore, IgM has the distinctive problem of being very difficult to be highly concentrated.
Clin. Chem. Lab. Med. 2000; 38(8): 759-764 (Non-Patent Document 3) and Journal of Immunological Methods, 111 (1988) 17-23 (Non-Patent Document 5) reported that IgM precipitates at a low salt concentration, and redissolves at a high salt concentration in a phosphate buffer and Tris-hydrochloride buffer that are weakly alkaline. Clin. Chem. Lab. Med. 2000; 38(8): 759-764 (Non-Patent Document 3) reports that, near pH 5, IgM readily precipitates and is difficult to handle, suggesting a pessimistic outlook for IgM solutions in weakly acidic buffers. This report thus gives no indication of the possibility of providing a highly concentrated IgM solution as a pharmaceutical or a bulk drug substance. This document also reports that when human sera comprising a high concentration of IgM are diluted with water, insoluble aggregates are generated as euglobulin precipitates, increasing the turbidity of the solution; but when the salt concentration is then raised by adding NaCl, Arginine, or such, the euglobulin precipitates redissolve. However, this report relates to the reconstitution of euglobulin precipitates, and does not provide any disclosures relating to suppression of increase in water-soluble aggregates of IgM. Furthermore, since patient-derived unpurified sera comprising various serum proteins are used in this document, the resulting insoluble aggregates may comprise proteins other than IgM. The effects on IgM solution in the absence of the other proteins are not described.
In Journal of Immunological Methods, 111 (1988) 17-23 (Non-Patent Document 5), a buffer comprising 0.1 M Tris-HCl and 1 M NaCl (pH 8) is used to redissolve euglobulin precipitates. However, the resulting recovery rate of IgM varies from 40% to >90% depending on antibodies or batch, indicating a low reproducibility. Additionally, although the Methods section describes that 5 to 10 mg/mL of purified antibodies were stored at 4° C. and −20° C., the Results section only describes that the antibodies could be stored for a few months at −20° C. without loss of function, and does not mention anything regarding storage at 4° C. or higher, at which temperature it is usually difficult to ensure stability. Accordingly, this report suggests the difficulty of reproducing precipitate reconstitution and the difficulty of ensuring stability during storage, when trying to provide a highly concentrated solution of IgM as a pharmaceutical product or a bulk drug substance.
BIOTECHNOLOGY 1993, 11, 512-515 (Non-Patent Document 4) and Journal of Immunological Methods, 111 (1988) 17-23 (Non-Patent Document 5) also describe the reconstitution of insoluble aggregates of antibodies as euglobulin precipitates, but the solubility is 10 mg/mL or less, indicating low solubility of IgM. There is no description at all regarding the stabilization of water-soluble aggregates.
Pharmaceutical Research 1994, 11(5), 624-632 (Non-Patent Document 6) discloses stabilization of IgM by PVP addition, but does not disclose the stabilization of highly concentrated antibodies. Journal of Immunological Methods 1995, 181(1), 37-43 (Non-Patent Document 7) discloses lyophilized formulations produced by addition of trehalose, but in this report, the antibody stability is insufficient and there is no description relating to stabilization of highly concentrated antibodies.    Patent Document 1: WO 2002/096457    Non-Patent Document 1: Proc. Natl. Acad. Sci. U.S.A, (1984) 81:6851    Non-Patent Document 2: Nature (1986) 321:521    Non-Patent Document 3: Clin. Chem. Lab. Med. 2000; 38 (8):759-764    Non-Patent Document 4: BIOTECHNOLOGY 1993, 11, 512-515    Non-Patent Document 5: Journal of Immunological Methods, 111 (1988), 17-23    Non-Patent Document 6: Pharmaceutical Research 1994, 11(5), 624-632    Non-Patent Document 7: Journal of Immunological Methods 1995, 181, 37-43    Non-Patent Document 8: Cytotherapy, 2001, 3(3), 233-242